In Vitro Cytotoxic Effects of Celecoxib, Mefenamic Acid, Aspirin and Indometacin on Several Cells Lines

Statement of the Problem Use of cyclooxygenase inhibitors as chemotherapy agents has attracted the attention of a large number of investigators in recent years. Given the importance of cancer therapy, only a limited number of studies have been carried out to investigate the effects of cyclooxygenase inhibitors on specific cell lines. Purpose This research aimed to determine the in vitro cytotoxic effects of cyclooxygenase inhibitors (COX-1 and COX-2 inhibitors) on KB, Saos-2, 1321N, U-87MG, SFBF-PI 39 cell lines. Materials and Method Powders of celecoxib, mefenamic acid, aspirin and indometacin were dissolved in the appropriate solvent. The viability of cell lines was carried out by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay technique. Data gathered from four separate experiments were expressed as mean±SD. Statistical significance was defined at p< 0.05 by using analysis of variance. Significant treatment mean values were subjected to post-hoc Tukey’s test. Results Celecoxib showed marked cytotoxic effects on KB, Saos-2, and 1321N cells, which was significant in comparison with the control group. Celecoxib was not effective in killing U-87MG cell line. Mefenamic acid exerted cytotoxic effects on KB, Saos-2, and 1321N cells, where the viability was approximately 75%. U-87MG cells were resistant to mefenamic acid. Indometacin had the highest rate of activity on U-87MG cells, which was significant in comparison with the control group. Aspirin did not exhibit any activity on these cell lines and was not effective in killing U-87MG, KB, Saos-2, and 1321N cells. Conclusion This research showed that celecoxib, indometacin, and mefenamic acid have the cytotoxic effects on KB, Saos-2, 1321N and U-87MG cell lines. Therefore, it appears that these drugs can be considered as anti-neoplastic agents in the experimental phase.


Introduction
In the United States, cancer is the second leading cause of death after cardiovascular diseases. In many developing countries, the incidence of cancer is much lower most likely because of the higher death rates due to infectious diseases or traumatic injuries. The studies shows that incidence of cancer is expected due to the increases in life expectancy, increasing proportion of elderly people, and successful control of childhood diseases. [1] At present, cancer and its treatment are considered as global problems. Complete removal of cancer without damage to the rest of the body is the goal of treatment, which sometimes can be accomplished by surgery. However, the propensity of cancers to invade the adjacent tissue or to spread to distant sites by microscopic metastasis often limits its effectiveness. Surgery often entails the removal of a wide surgical margin or a free margin. Radiation can also cause damage to normal tissues. [2] Chemotherapy is one of the most common treatment modalities for cancer. Based on the results of various researches, many chemotherapy agents have some side effects. Therefore, discovering new drugs, new properties of the existing drugs with lower side effects, and better treatment results is of great importance. [3] Some of the most promising pharmaceutical agents described to date for the prevention of cancer are the nonsteroidal anti-inflammatory drugs (NSAIDs). [4] These drugs are primarily used to treat pain and inflammation associated with arthritis. [5] NSAIDs inhibit the cyclooxygenase (COX) activity, and are also referred to as "COX inhibitors". There exist two isoforms of COX, with distinct tissue distributions and physiological functions. COX-1 is expressed in many tissues and plays a role in production of prostaglandins that control the normal physiological processes. On the other hand, COX-2 is pro-inflammatory in nature and is expressed only in response to certain stimuli such as mitogens, cytokines, and growth factors. [6] In vitro, in vivo, and observational evidence has demonstrated over-expression of celecoxib in solid malignancies including colon, prostate, breast, pancreas, lung, bladder, endometrium, skin basal membrane, and squamous cell malignancies. A significant relation has been established between over-expression of COX-2 and survival of patients with various cancers in retrospective studies. [4][5][6][7] Woo et al. reported that mefenamic acid had an inhibitory effect on proliferation of human liver cancer cells, and induced apoptosis in them. [7] Some epide-miological studies showed that intake of aspirin decreased the risk of developing esophageal carcinoma up to 90%. [5][6]8] Some other studies revealed that indometacin impaired the development and growth of head and neck squamous cell carcinoma (HNSCC) in experimental tumor models. [9] Given the importance of cancer therapy and the fact that only a limited number of studies have been carried out about the effects of cyclooxygenase inhibitors on these cell lines, the aim of this research was to determine the cytotoxic effects of cyclooxygenase inhibitors (COX-1 and COX-2 inhibitors) on KB (oral squamous cell carcinoma), Saos-2 (osteogenic sarcoma), 1321N (human brain astrocytoma), SFBF-PI 39 (Fibroblast-like from sheep brain) and U-87MG (malignant gliomas) cell lines in vitro.

Cell culture and cell lines
This study used KB, Saos-2, 1321N, U-87MG cell lines, which were purchased from the National Cell Bank of Iran (NCBI). These cells were immersed in RPMI 1640 medium containing 10% heat-inactivated fetal calf se-

Results
In this research, cytotoxic effects of four non-steroidal anti-inflammatory drugs were examined on KB, Saos-2, 1321N, SFBF-PI 39, and U-87MG cell lines. Table 1 shows the effects of celecoxib on the viability of cancer Indometacin had the highest rate of activity on U-87MG cells, which was significant in comparison with  Several studies have revealed that NSAIDs can even reverse the progress of some cancers. These authors believe that inhibitors of COX-1 and COX-2 can bring about a 50-93% reduction in the incidence of colorectal, prostate, lung, liver, and breast cancers. [12] Studies have also shown that these inhibitors can increase life span of people with breast, digestive system and lung cancers. [13][14] Research shows that shown that these drugs might be of benefit against the development and growth of malignancies. [18] This research showed that celecoxib and mefenamic acid had significant cytotoxicity on KB, Saos-2 and 1321N cell lines, even in low concentrations. Meanwhile, U-87MG cells were resistant to celecoxib and mefenamic acid. Indometacin had the highest rate of activity on U-87MG cells.
Wang et al. [19] showed that non-cytotoxic level of Indometacin reduced the cell invasion of malignant gliomas mediated by matrix metalloproteinases (MMP-2 and MMP-9). It also lowered down the activity of MMP-2 and MMP-9, and decreased the MMP-2 secre-tion of cell lines; while, it is reported that celecoxib could not induce significant autophagy in U87MG cell. [20] Liu et al. [21] were the first to describe tumor induction capacity of COX-2 over-expression. Celecoxib (COX-2 inhibitor) was used against colon carcinogenesis for the first time by Kawamori et al. [22] Matthias et al. [23] showed that defragmentation of DNA chains in cancerous and pre-cancerous cells decreased significantly when celecoxib was used. They concluded that this inhibitor was better than most other drugs because of its fewer side effects.
The Clinical investigations by Kliachkin et al. [26] showed that mefenamic acid decreased the activity of cathepsin D-like protease in cancerous colon tissue. The acid failed to affect the proteolytic activity of normal mucosa.
The results of this study showed that aspirin has no significant cytotoxic effects on cell lines, even in high concentrations. Kune et al. [29] reported that individuals who took aspirin regularly had 40% lower risk of colon cancer compared to those who did not. Some other epidemiological studies indicated that the relative risk of developing colon carcinoma was significantly lower (about 40-50%) in patients taking aspirin or other NSAIDs. [5] Another study demonstrated that regular daily use of aspirin was associated with 66% reduction in prostate cancer risk. [30] However, some studies demonstrated the opposite. Atula et al. [15] and Jaeckel et al. [31] showed that aspirin did not have any inhibitory effect on cancerous cells and it thinned the blood of patients who undergo chemotherapy, posing some problems for them.

Conclusion
This research showed that celecoxib and mefenamic acid have the highest cytotoxic effects on KB, Saos-2, 1321N, and U-87MG cell lines. Furthermore, indometacin has the highest cytotoxic effects on U-87MG cell line. Therefore, it appears that these drugs can be introduced as anti-cancer drugs in experimental phase.